Adapting virus filtration to continuous processing: Effects of product and process variability on filtration performance.

Virus filtration (VF) is an important unit operation in the manufacture of biotherapeutics that provides robust removal of potential virus contaminants. Small virus removal can be impacted by the low operating pressures and potential depressurization events that are often associated with continuous operations where increased operational flexibility for higher loading at low flux and low pressure is required. In this study, we evaluated the impact of low flux (7 LMH) and pressure interruptions on minute virus of mice (MVM) removal. We used long-term filtrations conducted to a target throughput of 1000 L/m2 with four different monoclonal antibodies on small-scale hollow fiber virus filters with a hydrophilic modified polyvinylidene fluoride membrane. These conditions are certainly challenging for any VF operation and ensuring robust viral clearance under such conditions is critical to the design and implementation of continuous VF. Planova BioEX filters effectively removed MVM at 4 log or greater when run continuously for up to 6 days. Interestingly, pressure increases associated with filter fouling over the duration of long-term filtrations were shown to be reflective of load material variability and could be remediated by implementation of an inline prefilter. Pressure interruptions had minimal impact on overall MVM logarithmic reduction value. Effective virus removal was achieved with pressure increases being largely product-specific, which demonstrates the capability of the virus filter to remove virus independent of pressure increases that are expected to occur with increased protein load.

Variables Impacting Silicone Oil Migration and Biologics in Prefilled Syringes.

Prefilled syringes (PFS) as a primary container for parenteral drug products offer significant advantages, such as fast delivery time, ease of self-administration and fewer dosing errors. Despite the benefits that PFS can provide to patients, the silicone oil pre-coated on the glass barrels has shown migration into the drug product, which can impact particle formation and syringe functionality. Health authorities have urged product developers to better understand the susceptibility of drug products to particle formation in PFS due to silicone oil. In the market, there are multiple syringe sources provided by various PFS suppliers. Due to current supply chain shortages and procurement preferences for commercial products, the PFS source may change in the middle of development. Additionally, health authorities require establishing source duality. Therefore, it is crucial to understand how different syringe sources and formulation compositions impact the drug product quality. Here, several design of experiments (DOE) are executed that focus on the risk of silicone oil migration induced by syringe sources, surfactants, protein types, stress, etc. We utilized Resonant Mass Measurement (RMM) and Micro Flow Imaging (MFI) to characterize silicone oil and proteinaceous particle distribution in both micron and submicron size ranges, as well as ICP-MS to quantify silicon content. The protein aggregation and PFS functionality were also monitored in the stability study. The results show that silicone oil migration is impacted more by syringe source, siliconization process and surfactant (type & concentration). The break loose force and extrusion force across all syringe sources increase significantly as protein concentration and storage temperature increase. Protein stability is found to be impacted by its molecular properties and is less impacted by the presence of silicone oil, which is the same inference drawn in other literatures. A detailed evaluation described in this paper enables a thorough and optimal selection of primary container closure and de-risks the impact of silicone oil on drug product stability.

Development of a transient inline spiking system for evaluating virus clearance in continuous bioprocessing-Proof of concept for virus filtration.

The development of continuous/connected bioprocesses requires new approaches for viral clearance validation, both for specific unit operations and for the overall process. In this study, we have developed a transient inline spiking system that can be used to evaluate virus clearance at distinct time points during prolonged operation of continuous bioprocesses. The proof of concept for this system was demonstrated by evaluating the viral clearance for a virus filtration step, both with and without a prefilter upstream of the virus filter. The residence time distribution was evaluated using a previously identified noninteracting fluorescent tracer, while viral clearance was evaluated from measurements of the virus titer in samples obtained downstream of the virus filter. The measured log reduction values (LRV) for ϕX174, minute virus of mice, xenotropic murine leukemia virus, and a noninfectious mock virus particle were all within 0.5 log of those obtained using a traditional batch virus challenge for both model and real-world process streams (LRV between 2.2 and 3.4 for ϕX174 using a single layer of virus filter). The results demonstrate the effectiveness of transient inline spiking to validate the virus clearance capabilities in continuous bioprocessing, an essential element for the adoption of these processes for products made using mammalian cell lines.

An innovative strategy to recycle permeate in biologics continuous manufacturing process to improve material efficiency and sustainability.

Intensified perfusion processes are an integral part of continuous manufacturing for biopharmaceuticals which enable agile operations and significant reduction in cost of goods. However, they require large volumes of media to support robust cell growth and maintain high productivity, posing substantial challenges to operations, logistics, and process sustainability. This study explores a novel strategy for reprocessing and reusing permeate from perfusion cultures for mAb production. The concept was initially evaluated by recycling permeate, Protein A flow-through (ProA FT) and CEX processed ProA FT in deep-well plate mock perfusion and ambr® 250 perfusion formats. Further processing of ProA FT through a cation exchange depth filter before recycling reduced process impurities such as host cell proteins (HCPs) and DNA. However, a direct replacement of fresh media with spent media reduces nutrient depth which results in a concomitant reduction in productivity. In ambr® 250 bioreactors, recycling of ProA FT at 25%-50% replacement rates (defined as the fraction of recycled material in media) resulted in a 13%-30% reduction in cumulative productivity while maintaining product quality. To mitigate this, we used media concentrates which allowed independent modulation of media depth by replacing a portion of diluent WFI with recycled material. Results from deep-well mock perfusion studies demonstrated that comparable or higher productivities relative to control can be achieved with this approach. Taken together, our study demonstrates the feasibility of recycling permeate in perfusion cultures. Process mass intensity (PMI) calculations reveal that this approach can meaningfully improve material efficiency by reducing water consumption, thereby enhancing overall bioprocess sustainability.

Considerations for Implementation of a New Drug Substance Container for Subzero Storage.

Therapeutic manufacturing has become globalized in recent decades, necessitating transportation of drug substance across the world. The outcome of this expansion is significant costs for shipment and added risk of damage to the drug substance containers. There are multiple container options with various materials of construction for storage of Biologics drug substance (DS). This study evaluates a newly designed CryoVault™ container and previously characterized CelsiusPak® bag container using a well-represented scale-down model. Consideration of an appropriate storage container includes the risk assessment of the design and material of construction, which can potentially impact product quality attributes, stability and container leachables. An extensive data package, including product stability over time and temperature with respect to impact of extractables and leachables from different containers undergoing a typical one freeze/thaw cycle process was evaluated. This drove to the decision for implementation of a container into the drug substance manufacturing process.

A direct RT qPCR method for quantification of retrovirus-like particles in biopharmaceutical production with CHO cells.

Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing biologic drugs, like monoclonal antibody, in the biopharmaceutical industry. Retrovirus-like particles (RVLPs) are made during the manufacturing process with CHO cells and it is incumbent upon the manufacturer to perform risk assessment based on levels of RVLP in unprocessed bulk. Quantification of RVLP using electron microscopy (EM) is the standard method. However, reverse transcription based real-time PCR (RT qPCR) is an alternative method available. This method involves RNase digestion of cell culture fluid to remove free RNA, followed by extraction of total nucleic acid and digestion with DNase to remove extracted DNA molecules, and then finally reverse transcription and PCR. Here we report a method where the nucleic acids extraction step is eliminated prior to qPCR. In this method the cell-free culture supernatant sample is digested with thermolabile DNase and RNase at the same time in a 96-well PCR plate; subsequently the enzymes are heat-denatured; then RT qPCR reagents are added to the wells in the PCR plate along with standards and controls in other wells of the same plate; finally the plate is subjected to RT qPCR for analysis of RVLP RNA in the samples. This direct RT qPCR method for RVLP is sensitive to 10 particles of RVLP with good precision and accuracy and has a wide linear range of quantification. The method has been successfully tested with different production batches, shown to be consistent, and correlates well with the extraction-based method. However, the results are about 1-log higher compared to EM method. This method simplifies the RVLP quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods. Additionally, the method can be an added tool for viral clearance studies, by testing process-intermediate samples like Protein A column and ion-exchange column eluates, for increased confidence in purification of biologics manufactured in CHO cell culture.

Quantitative assessment of environmental impact of biologics manufacturing using process mass intensity analysis.

Process mass intensity (PMI) is a benchmarking metric to evaluate the efficiency of a manufacturing process, which is indicative of the environmental impact of the process. Although this metric is commonly applied for small molecule manufacturing processes, it is less commonly applied to biologics. In this study, an Excel based tool developed by the ACS GCI Pharmaceutical Roundtable was used to calculate PMI of different manufacturing processes for a monoclonal antibody (mAb). For the upstream process, three different versions were compared: fed-batch, fed-batch with N-1 perfusion, and perfusion in the N-stage bioreactor. For each upstream process version, an appropriate downstream operational mode was evaluated from the following: a column chromatography process utilizing Protein A and anion exchange (AEX) resin, a Protein A column and an AEX membrane, and a three-column periodic counter-current (3C PCC) chromatography process for Protein A and an AEX membrane. The impact of these different process variations on PMI was evaluated. Of all the process inputs, water contributes about 92-94% of the overall PMI. Additionally, the upstream processes and the chromatography steps account for 32-47 and 34-54% of the overall PMI, respectively. Sensitivity analysis was performed to identify opportunities for further reducing PMI. These data indicate that a semicontinuous manufacturing process (perfusion, 3C PCC, and AEX membrane) is the most efficient process, resulting in a 23% reduction of PMI when compared with the fed batch and two-column chromatography process. Together, PMI can be used to guide the development of efficient and environmentally sustainable mAb manufacturing processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1566-1573, 2018.

Impact of Freeze/Thaw Process on Drug Substance Storage of Therapeutics.

The storage of drug substance at subzero temperatures mitigates potential risks associated with liquid storage, such as degradation and shipping stress, making it the best solution for long-term storage. However, slower (generally uncontrolled) rates of freezing and thawing of drug substance in conventional large storage containers (>2L) can lead to greater cryoconcentration (exclusion of solute molecules) resulting in zones of higher protein and excipient concentrations and changes to the desired formulation pH and excipient concentration. These conditions can negatively impact product quality, thus changing the target product profile. Freeze/thaw studies can provide valuable knowledge on the molecule even when performed from an early formulation image. This study attempts to provide guidance and strategy for planning of drug substance freeze and thaw studies in early development using a scale-down model, evaluating the impact of the (1) freeze/thaw rate, (2) mode of freezing, (3) drug substance container, (4) drug substance concentration, and (5) formulation on the drug substance product quality. Data presented in this study showed no impact on drug substance product quality after undergoing the typical one freeze/thaw cycle process for the variables evaluated. These findings suggest that a qualified scale-down model is not required for early phases of process development and that existing small-scale models can be used for drug substance storage development studies. Based on our experience, a workflow is suggested with minimal experimental design to reduce the material requirement by >70% at early stages of product development to reduce constraints.

Prediction of viral filtration performance of monoclonal antibodies based on biophysical properties of feed.

Controlling viral contamination is an important issue in the process development of monoclonal antibodies (MAbs) produced from mammalian cell lines. Virus filtration (VF) has been demonstrated to be a robust and effective clearance step which can provide ≥4 logs of reduction via size exclusion. The minimization of VF area by increasing flux and filter loading is critical to achieving cost targets as VFs are single use and often represent up to 10% of total purification costs. The research presented in this publication describes a development strategy focused on biophysical attributes of product streams that are directly applicable to VF process performance. This article summarizes a case study where biophysical tools (high-pressure size exclusion chromatography, dynamic light scattering, and absolute size exclusion chromatography) were applied to a specific MAb program to illustrate how changes in feed composition (pH, sodium chloride concentration, and buffer salt type) can change biophysical properties which correlate with VF performance. The approach was subsequently refined and expanded over the course of development of three MAbs where performance metrics (i.e., loading and flux) were evaluated for two specific virus filters (Viresolve Pro and Planova 20N) during both unspiked control runs and virus clearance experiments. The analyses of feed attributes can be applied to a decision tree to guide the recommendation of a VF filter and operating conditions for use in future MAb program development. The understanding of the biophysical properties of the feed can be correlated to virus filter performance to significantly reduce the mass of product, time, and costs associated with virus filter step development.

The impact of sialic acids on the pharmacokinetics of a PEGylated erythropoietin.

Erythropoietin (EPO) is an important molecule in the erythropoiesis and various forms of EPO have been marketed in managing anemia in humans. Long acting EPOs for less frequent dosing have been generated either by increasing the number of glycosylation sites of the EPO molecule or by linking it to a polyethylene glycol (PEG). We have generated recombinant human EPO (rhEPO) using glycoengineered Pichia pastoris strains and evaluated the pharmacokinetics (PK) in rats of this molecule linked to a 40 kDa PEG (PEGylated rhEPO), in relation to its glycosylation patterns. As expected, the PEGylated rhEPO exhibited a significant improvement in half-life of serum when compared with the non-PEGylated version. Interestingly, the PK properties of the PEGylated rhEPO molecule were also significantly influenced by the glycosylation profile. Specifically, PEGylated rhEPO with a significantly higher sialic acid content in the biantennary structure (high A2) exhibited lower systemic clearance and higher systemic exposure than those with a lower sialic acid content (low A2) following either intravenous or subcutaneous administrations. These results suggest that A2 content may be one of the important criteria for release in manufacturing PEGylated rhEPO to ensure consistent PK.